Following Cell Lineages
Studying embryos at the level of the cell lineage has been possible because of the transparency of C. elegans at all stages. During characterization of wild-type development, lineages were originally followed with repeated observations by differential interference microscopy, aided by some video recordings for early stages (Sulston et al.,1983). Subsequently, time-lapse four-dimensional (4D) microscopy has allowed partial lineages to be constructed retrospectively in single embryos until muscle movements commence at approximately ∼400 min after first cleavage, at the 1.5-fold stage (Schnabel et al.,1997; Chisholm and Hardin,2005). Early technologies used analog laserdisc video of differential interference contrast microscopy (Hird and White,1993; Schnabel et al.,1997). More recently, computer-assisted fluorescence time-lapse microcopy using transgene reporters has been developed (discussed below) (Murray et al.,2006). In both cases, lineage reconstruction can be facilitated by computer software such as Simi BioCell or StarryNite (Schnabel et al.,1997; Bao et al.,2006; Murray et al.,2006).
Studying cell fate at this level can is important for understanding the nature of changes in cell fate specification that result from experimental perturbation. As mentioned above, blastomeres that have undergone a change in fate continue to divide but ultimately give rise to different tissues. In the process, the transformed lineage may adopt a cell division pattern characteristic of a new lineage. For example, an apparently complete transformation is visible in the case of deletions that remove end-1 and end-3, a pair of linked GATA factor genes that together specify the endoderm fate (Zhu et al.,1998; Maduro et al.,2005a). In embryos lacking function of both genes, E adopts the fate of C, producing body muscle and epidermis, with a transformation of its lineage to one that is C-like. However, such changed fates do not always show a clear lineage transformation. In mex-1 mutant embryos, the AB grand-daughters adopt MS-like fates, but the transformations in the lineage itself are not as clear (Mello et al.,1992; Schnabel et al.,1996). Presumably, incomplete transformations in mutant backgrounds result from competition among multiple factors in the same lineage, as well as a change in cell–cell interactions when a blastomere adopts a fate outside of its normal context.
Because of the mosaic nature of C. elegans embryonic development, in practice it is not necessary to evaluate the cell division pattern of an entire lineage to identify a cell fate transformation. Ectopic activation of early GFP reporters that anticipate the descendants made in a particular lineage can be taken as supportive evidence of a transformation in fate. At the end of embryogenesis, the location and number of ectopically produced cells can be used to suggest a cell fate transformation through the absence of a particular tissue and the excess of another. This is usually done through the light microscope, but transmission electron microscopy has also been used to characterize ectopically mis-specified tissues (Wolf et al.,1983; Bowerman et al.,1992). A more confident assignment of ectopic tissues to an early progenitor can be confirmed by using a laser microbeam to ablate all other blastomeres, as was done to characterize transformed E and MS blastomeres in end-1,3(−), med-1,2(RNAi), and tbx-35(−); ceh-51(−) embryos (Zhu et al.,1997; Maduro et al.,2001; Broitman-Maduro et al.,2009). Cell morphology, antibody staining or green fluorescent protein (GFP) reporters can then be used to identify the descendants produced in the resultant partial embryo.
Examining the Influence of Cell–Cell Interactions
One of the more elegant techniques in the C. elegans embryo is the study of contextual effects on specification by direct manipulation of blastomeres participating in a cell–cell interaction. The study of context-specific effects on cell fate can be achieved without breaching the eggshell, by putting pressure from the outside to nudge cells, as was used to characterize interactions that make ABp different from ABa, and in the study of left–right asymmetry (Priess and Thomson,1987; Wood,1991). A laser microbeam can be used to ablate particular cells and prevent cell–cell interactions, as was done to characterize specification within the AB lineage (Gendreau et al.,1994) or to block generation of AB- or MS-derived pharynx (Hunter and Kenyon,1996). To isolate or block specific cell–cell interactions, cells can be extruded through holes in the eggshell to reduce the number of possible cell contacts in the remaining cells (Priess and Thomson,1987). Alternatively, particular cell–cell interactions can be reconstructed outside the eggshell, as was done to characterize gut specification (Goldstein,1992). Cell–cell contacts up to the 150-cell stage were recently characterized systematically in intact embryos (Hench et al.,2009). In that study, approximately 1,500 cell–cell contacts lasting 2.5 min each were identified, and the cell interactions were modeled by computer. This resource, called Ce2008, will likely be very useful for future studies on cell interactions (Hench et al.,2009).
Forward and Reverse Genetics
In addition to embryo manipulations, cell–cell interactions can also be studied genetically. C. elegans possess a hermaphrodite mode of reproduction, which has greatly facilitated the recovery of mutations in developmentally important genes. The first regulators to be identified that affected early embryonic cell fate specification events were recovered in forward mutagenic screens that looked for maternal-effect embryonic lethal phenotypes exhibiting a deficit of tissues of one type and an excess of another. Chemical or transposon-mediated mutagenesis screens, followed by mapping and molecular cloning, have proven to be good ways of identifying such regulators (Priess et al.,1987; Kemphues et al.,1988). Such screens recovered glp-1, which encodes a Notch-like receptor important for asymmetries in the AB lineage; apx-1, which encodes a Delta-like ligand that mediates a GLP-1-dependent cell–cell interaction between P2 and ABp that alters the fate of ABp (Mango et al.,1994); a maternal-specific allele of pop-1, which encodes the C. elegans TCF-like Wnt effector, important for MS and E fates (Lin et al.,1995; Shetty et al.,2005); and skn-1, important for specification of EMS fate (Priess et al.,1987; Bowerman et al.,1992). Similar zygotic screens identified the two endoderm specification genes end-1 and end-3, by large deletions that removed both genes (Zhu et al.,1997; Maduro et al.,2005a), the epidermal specification gene elt-1 (Page et al.,1997), and tbx-37 and tbx-38, which participate in specification of some ABa descendants (Good et al.,2004).
Factors not accessible by forward mutagenesis, for example due to redundancy or requirement of genes in other processes, can be discovered by other approaches. Regulators can be identified by their expression in an early blastomere lineage. pal-1, which encodes a Caudal-like homeoprotein, was first identified by zygotic mutants and later found to play a central role in specification of C and D, the somatic descendants of P2, following the discovery that PAL-1 protein is found in the early P2 and EMS lineages (Hunter and Kenyon,1996; Hunter et al.,1999). Similarly, tbx-35 was identified as a candidate MS factor because of its expression in the early MS lineage (Robertson et al.,2004; Broitman-Maduro et al.,2005).
The discovery of RNA interference (Fire et al.,1998), coupled with the completion of the C. elegans genome sequence (Consortium,1998), led to the use of reverse genetics as a method to identify factors important for cell specification. Components of the Wnt/MAPK signaling pathway that mediate the P2-EMS interaction were identified by both genetics and RNAi (Rocheleau et al.,1997; Thorpe et al.,1997). A systematic search of GATA-like transcription factors encoded in the C. elegans genome led to the identification of the MED-1,2 divergent GATA factors, which specify MS and participate in specification of E as first characterized by RNAi (Maduro et al.,2001; Goszczynski and McGhee,2005). More recently, a genome-wide RNAi screen identified ceh-51, a gene which functions in MS specification (Broitman-Maduro et al.,2009). Although originally thought to act in muscle specification because of its larval arrest phenotype, the expression of ceh-51 was found to occur in the early MS lineage, directly downstream of the MS specification factor TBX-35 (Broitman-Maduro et al.,2006,2009).
As another reverse genetics approach, candidate genes may be selectively targeted for recovery of chromosomal mutations. Gene knockouts made following UV/TMP mutagenesis (Gengyo-Ando and Mitani,2000) are available from the National Bioresource Project (Mitani laboratory, Japan) and the Caenorhabditis Genetics Center (CGC, University of Minnesota, MN). Mutants at the CGC are made by the C. elegans Gene Knockout Consortium (Vancouver, British Columbia, Canada, and Oklahoma City, OK; http://celeganskoconsortium.omrf.org). Random insertions of transposons, such as Tc1 and Mos1, have also been generated (Zwaal et al.,1993; Granger et al.,2004). Alleles generated in these screens have been used to study chromosomal mutants for MS and E specification factors (Broitman-Maduro et al.,2006,2009; Maduro et al.,2007). For example, after the T-box factor gene tbx-35 was identified as a candidate for MS specification because of its early MS lineage expression, a deletion allele, created by reverse genetics, was used to characterize its loss-of-function phenotype (Robertson et al.,2004; Broitman-Maduro et al.,2005,2006). The chromosomal mutant proved invaluable for identification of tbx-35 as a critical MS regulator, as RNA interference against tbx-35 was found to be almost completely ineffective (Broitman-Maduro et al.,2006). RNAi against the related genes tbx-37 and tbx-38, which function together in specification of ABa descendants, was also found to be ineffective (Good et al.,2004), suggesting that early zygotic regulator genes may be generally resistant to RNAi.
Before the establishment of techniques to make C. elegans transgenic, identification of cells or tissue types was achieved primarily by their appearance in the light microscope or by immunohistochemistry (Duerr,2006; Yochem,2006). RNA in situ hybridization has also been used to localize transcripts (e.g., Fig. 2A; Seydoux and Fire,1995; Kohara,2001; Coroian et al.,2005).
Use of reporter transgenes, by far the most frequently used method to assess gene expression and to mark tissues in C. elegans, became standardized through the use of gonadal microinjection, first with lacZ as a reporter, and subsequently GFP and its variants (Fire et al.,1990; Mello et al.,1991; Chalfie et al.,1994). Transgene arrays made this way are generally useful for zygotic genes, but not for maternal genes which frequently undergo silencing due to the repetitive nature of the arrays (Kelly et al.,1997). Problems with expression of maternally expressed transgenes and mosaic expression of zygotic transgenes have been overcome by the use of complex arrays (Kelly et al.,1997), microparticle bombardment (Praitis et al.,2001), or transposon-mediated chromosomal integration (Frokjaer-Jensen et al.,2008). The latter approach, which relies on transgene-dependent repair of a chromosomal Mos1 excision event, is advantageous as it results in a known location of the inserted transgene, favors low copy number insertions, and requires no additional technology to implement in a lab that already has standard microinjection working (Frokjaer-Jensen et al.,2008).
Determining which cells in the early embryo express a given gene is usually a matter of recognizing the expressing cells by their morphology and context (e.g., end-3 reporter expression in Fig. 2B, hlh-1 reporter expression in Fig. 2H). For more complex patterns, cell identification can be facilitated by comparison with 4D time lapse recordings (Hope,1994). Alternatively, mutant backgrounds that transform specific lineages can be used to confirm that expression patterns change appropriately (Maduro et al.,2001). More recently, semi-automated 4D fluorescence microscopy has been used to follow expression of embryonic transgenes simultaneously with the lineage (Murray et al.,2008). In these experiments, the expression of a cell-specific reporter is viewed in a separate fluorescent channel from a ubiquitously expressed nuclear reporter, with the pattern of cell divisions informing the cell identities. Recently, a computational approach has been developed to analyze expression of genes in L1 larvae (Long et al.,2009). In this approach, cells are identified by their positions in the larva, rather than their lineage. Using this method, single-cell resolution expression patterns of 93 reporter genes were used to deduce the points in the embryonic cell lineage that particular tissues were specified by asymmetric cell divisions (Liu et al.,2009). The coupling of fluorescent in vivo reporters with computer-aided image analysis is clearly emerging as a powerful approach for the rapid characterization of factors that are involved in specification of cells or tissues.
Aside from the use of transgene reporters to identify the cells in which a factor is expressed, there are three other applications of transgenes that are worth mentioning. First, factors that are hypothesized to act as “master genes” can be tested outside their normal context through overexpression driven by a heat shock promoter (Stringham et al.,1992; Horner et al.,1998; Zhu et al.,1998; Maduro et al.,2001; Broitman-Maduro et al.,2006; Fukushige et al.,2006). Such constructs permit the conditional activation of the factor of interest in essentially all somatic cells in the embryo (e.g., hs-tbx-35 embryo in Fig. 2I,J; Stringham et al.,1992). Second, translational GFP fusions can be used to visualize subcellular localization of tagged proteins (e.g., GFP-tagged END-3 in Fig. 2B). This has been done for proteins that show differential subcellular localization or stability in different cells, such as the β-catenin/asymmetry pathway components TCF/POP-1 (Maduro et al.,2002) and the divergent β-catenin SYS-1 (Huang et al.,2007; Phillips et al.,2007). A third transgene-based technique, the “nuclear spot assay”, takes advantage of the repetitive nature of multicopy transgene arrays. In this approach, a GFP-tagged transcription factor can be localized to the subnuclear region occupied by target arrays that contain its binding sites. For example, this approach was used to show interaction of the endoderm factor ELT-2 with its own promoter (Fukushige et al.,1999) and interaction of POP-1 and MED-1 with the promoters of end-1 and end-3 (e.g., GFP::MED-1 subnuclear spots in Fig. 2C; Maduro et al.,2002). In principle, this assay could be used to identify and map transcription factor-DNA interactions in an in vivo context, with no prior knowledge of the binding sites.
Studies of C. elegans benefit from a complete genome sequence with thorough annotation by means of the centralized resource WormBase (http://www.wormbase.org) (Consortium,1998; Harris et al.,2010). The genome sequence has greatly facilitated molecular identification of mutated genes and has also been used for multiple other applications. Owing to the ability of C. elegans to knock down endogenous gene expression by RNAi through ingested dsRNA (Timmons and Fire,1998), libraries of bacteria have been constructed that express dsRNA targeted to each gene, permitting genome-wide RNAi-based screens by bacterial feeding (Kamath and Ahringer,2003; Johnson et al.,2005). A comprehensive list of the entire suite of ∼1,000 C. elegans transcription factors has been compiled, and this information has been used to generate GFP reporters for many of these (Reece-Hoyes et al.,2005,2007). Yeast two-hybrid studies have been used to identify protein–protein interactions (Boxem et al.,2008). The results of genome-wide screens, expression pattern studies and protein interactions become available on WormBase soon after publication.
The genome sequence has been invaluable for whole-genome studies of gene expression. An early use of the sequence was in the generation of microarrays, which can be used to identify genes expressed in various tissues such as the germline (Reinke et al.,2000). Microarrays were used to identify transcripts expressed in a small number of precisely staged early embryos (Baugh et al.,2003). To work with such small samples, protocols were developed to linearly amplify transcripts from limiting amounts of RNA (Eberwine,1996; Baugh et al.,2003). These approaches were used to identify genes that participate in C specification, by isolating mRNA from intact embryos in mutant backgrounds that either created extra C cells or abolished their specification (Baugh et al.,2005). These experiments elucidated a gene network for PAL-1–dependent cell specification of the early C lineage (Baugh et al.,2005). In a similar strategy designed to identify genes that function in a specific tissue, microarrays were used to identify 240 transcripts enriched in the pharynx in similarly made pharynx(+) and pharynx(−) embryos (Gaudet and Mango,2002).
Other genome-wide techniques are aimed at identifying cis-regulatory sites of interest. For example, all promoters, comprising the C. elegans “promoterome,” have been cloned and used to generate reporter GFP fusions of a subset of these (Dupuy et al.,2004,2007). A subset of protein–DNA interactions have been identified by yeast one-hybrid analysis using the promoterome (Deplancke et al.,2006). A recent study reported the use of chromatin immunoprecipitation as a method for verifying direct interaction between PAL-1 and the muscle regulator MyoD/hlh-1 (Lei et al.,2009). A large, multi-investigator project, modENCODE (model organism ENCyclopedia Of DNA Elements), is working to systematically document all transcription factor-DNA interactions in C. elegans and Drosophila (Celniker et al.,2009). Information from all of these genome-level projects will likely lead to identification of new genes and protein-DNA interactions that are important for cell specification.
Other approaches can be used to identify lineage-specific transcripts. These include using tissue-specific expression of epitope-tagged Poly-A binding protein to immunoprecipitate transcripts, coupled with microarray analysis or serial analysis of gene expression (SAGE) tagging (Roy et al.,2002; Pauli et al.,2006; McGhee et al.,2009). Newer massively parallel sequencing methods, such as RNA-seq, permit efficient identification of full-length transcripts by coupling the technology with approaches that enrich for mRNAs present in particular lineages or cells (Hillier et al.,2009; McGhee et al.,2009; Wang et al.,2009). The field is just beginning to explore the possibilities with this new technology.
Biochemical methods have been used to characterize protein-DNA or protein–protein interactions important for cell specification in C. elegans. As examples, DNA-binding properties of TCF/POP-1 and interactions of POP-1 with the Nemo-like kinase LIT-1 and the divergent β-catenin WRM-1 were studied in cultured cells (Shin et al.,1999; Korswagen et al.,2000); co-immunoprecipitations from C. elegans embryos were used to detect interaction between WRM-1 and LIT-1 (Rocheleau et al.,1999); gel shift analysis and DNaseI footprinting studies have been reported for E and MS specification factors in C. elegans (Stroeher et al.,1994; Hawkins and McGhee,1995; Broitman-Maduro et al.,2005,2009); mass spectrometry was used to identify proteins that can bind to the promoter of end-1 (Witze et al.,2009); and an NMR solution structure has been solved for the MED-1 DNA-binding domain (Lowry et al.,2009). Therefore, although it can be difficult in practice to obtain biochemical quantities of C. elegans embryos (particularly those of a specific stage), this has not been a severe limitation to doing biochemical experiments.
Comparative Studies With Other Nematodes
Approximately a dozen species have been identified in Caenorhabditis (Kiontke et al.,2004). Embryogenesis is found to be very similar between C. briggsae and C. elegans (Zhao et al.,2008). However, there are RNAi-induced phenotype differences between these two species with respect to the orthologues of skn-1 and pop-1 (Lin et al.,2009), suggesting that the underlying gene networks have undergone some changes. Hence, comparative studies of embryo cell specification among Caenorhabditis species may be a way to probe the flexibility of specification mechanisms that produce the same output, similar to what has been done postembryonically in the vulval lineages (Felix,2007). Genome sequences are available for different nematode species, and comparisons among noncoding regions have generated cisRED, a compendium of conserved putative cis-regulatory sites (Sleumer et al.,2009). RNAi by bacterial feeding is also possible in C. briggsae (Winston et al.,2007); this technique was used in examining knockdown of skn-1 and pop-1 (Lin et al.,2009), and it should be possible to perform genome-wide RNAi screens to identify cell fate genes in C. briggsae. A recent comparative study described differences in very early embryonic development, before cell specification events take place, among multiple nematode species in different genera (Brauchle et al.,2009). Comparisons of cell specification events can therefore be useful both from an evolutionary standpoint, and to inform studies of C. elegans development.
The C. elegans system clearly offers a rich set of tools for the study of cell fate specification pathways, and newer approaches that exploit massively parallel sequencing and computer-aided analysis of expression patterns will almost certainly revolutionize the next phase of work in this field. Many themes of development have already emerged from cell specification studies in C. elegans, as described below.